1. Field of Invention
A systematic method is presented for discovering and determining the suitable conformation of a mono-specific B-cell epitope appropriate for eliciting a protective antibody response.
2. Description of the Related Arts
Lack of definition of a mono-specific B-cell epitope The heterogenic antibody responses to complex protein antigens frequently lead to futile protective immune response. There is an urgent need for understanding the entity of protective antigen, and even the fine specificity, i.e., the mono-specific B-cell epitope of the protective antigen. To identify the mono-specific B-cell epitope as therapeutic target is important due to the plethora and complexity of B-cell epitopes in a complex biological macromolecule from pathogenic microbes, toxins, and cancer cells. In particular, suboptimal antibody responses to a protective B-cell epitope may ensue as a result of antigen competition and downregulation of response to this particular protective B-cell epitope due to presence of a myriad of other multiple B-cell epitopes.
Thus far, there does not exist a systemic method to first delineate such mono-specific B-cell epitopes, and second to conform and improve these B-cell epitopes for a high affinity interaction with antibodies. Most studies employ the overlapped synthetic peptide sequences, and couple each respective peptide to the carrier protein for raising mono-specific antibody. For decades, B-cell epitopes can not be simply synthesized and coupled onto carrier protein for studying their native antigenicity. Antibodies raised by this method while adequate for reactive with denatured proteins according to western blot analysis, are generally incapable of reacting with native proteins, and therefore are useless for potential higher impact for neutralizing toxins, microbes or cancerous cells.
Understanding and subsequently utilizing B-cell epitopes as well-defined vaccine can improve vaccine efficacies. Linear peptides synthesized from regions of protein, including solvent exposed loop region according to the X-ray structure of a protein, and coupled to immunogenic carrier protein for immunization, were known to induce antibodies that react with linear peptide or denatured protein but are not cross-reactive with the native protein.
Protein Scaffold and stability The desirable loop sequences spanning part of the scaffold protein are characterized by hydrophilicity in nature, surface-exposed, and mobile. It is known that foreign B-cell antigenic loops grafted in complementarity-determining regions (CDR) of the immunoglobulin scaffold, exhibit constrained antigenic conformations similar to those expressed as the native loop of parent molecules. This process of “antigenization of antibody” utilizes the immunoglobulin fold as a scaffold to constrain a grafted oligopeptide (Zanetti, Antigenized antibodies and genes, U.S. Pat. No. 5,583,202; Gerloni et al., 1997 Nat. Biotech. 15: 876; Xiong et al., 1997. Nature Biotech. 15:882). CDR regions of immunoglobulin are thermodynamically less resilient to inserted foreign determinants into the existing loop sequence with solubility affected. Although in most case immunoglobulin scaffold is successful for presenting the linear sequence-dependent epitope for inducing cytotoxic T-lymphocytes. The issue remains open as to the appropriate scaffolding protein for constraining conformation-dependent B-cell epitopes.
In addition to CDR3 of immunoglobulin scaffold, proteins of super-immunoglobulin gene family, i.e., CTLA4, fibronection domain have also been tested as protein scaffold (Skerra, 2000. Mol. Reg. 13:167; Binz and Pluckthun, 2005 Curr. Opin. Biotech. 16:459). These proteins exhibiting β-pleated sheet immunoglobulin fold, also display similar range of melting temperatures to that of immunoglobulin as a heat labile protein. Although CTL-A4 and fibronectin were employed for inserting an aptameric peptide library of random specificities in the loop region since only the conformationally viable species will participate in the selection process at the expense of pre-selection of library repertoire.
However for inserting a peptide of pre-determined sequence such as the loop sequences from the X-ray structure of a protein, each relevant sequence in each protein is unique and non-replaceable, and there is stringent demand for successfully constraining such unique pre-determined sequences. Therefore, a reliable thermostable protein scaffold that provides high probability of constraining capacity for pre-determined amino acid sequence becomes a necessity for antigenic systems-discovery platform.